SARS-CoV-2 Seropositivity in Urban Population of Wild Fallow Deer, Dublin, Ireland, 2020–2022

SARS-CoV-2 can infect wildlife, and SARS-CoV-2 variants of concern might expand into novel animal reservoirs, potentially by reverse zoonosis. White-tailed deer and mule deer of North America are the only deer species in which SARS-CoV-2 has been documented, raising the question of whether other reservoir species exist. We report cases of SARS-CoV-2 seropositivity in a fallow deer population located in Dublin, Ireland. Sampled deer were seronegative in 2020 when the Alpha variant was circulating in humans, 1 deer was seropositive for the Delta variant in 2021, and 12/21 (57%) sampled deer were seropositive for the Omicron variant in 2022, suggesting host tropism expansion as new variants emerged in humans. Omicron BA.1 was capable of infecting fallow deer lung type-2 pneumocytes and type-1–like pneumocytes or endothelial cells ex vivo. Ongoing surveillance to identify novel SARS-CoV-2 reservoirs is needed to prevent public health risks during human–animal interactions in periurban settings.

total number of persons that attempted to interact with the herd during the observation session, the day of the week (categorical: Friday, Saturday, Sunday), the time of day, the amount of time the observer spent monitoring the herd (i.e., sampling effort), the month of observation (categorical: June, July) and year of study (categorical: 2019, 2020, 2021) as explanatory variables in our model.All numerical predictors were scaled to improve model convergence and were included as both single and quadratic terms in the model to factor in nonlinear patterns.
We also included 2-way interactions in the model as follows: sex and age, sex of the deer and total number of persons attempting to interact with the herd, and sex and herd size, all of which were included in both their linear and quadratic forms leading to a total of 6 interactions.
Variable selection rationale and a priori expectations are detailed as previously described (1).All predictors included in the model structure were not collinear (Pearson's correlation coefficient rp<0.7)(3).We then extracted the random intercepts estimated by the generalized linear mixedeffects model for each individual ID, also known as conditional modes or best linear unbiased predictors (BLUPs) (4), which were highly repeatable across animals (1,5).This model constituted a ranking system, which ranged from lowest to highest deer begging rates after taking all model predictors into account.
Step 2, Extracting Begging Categories We extracted the begging ranks (i.e., random intercept or BLUP value) and calculated 95% CIs for each random intercept value for each deer ID and then summarized them into behavioral categories.The behavioral categories were identified depending on how the random effects and related CIs were distributed around the median random effect (zero, i.e., the median begging behavior of the population).Ultimately, begging behavior exists on a continuum, but we subdivided those behaviors into 3 categories for clarification: random effect 95% CI >0 (consistent beggars), 95% CI overlapped 0 (occasional beggars), or 95% CI <0 (rare beggars).This approach considers and categorizes the BLUPs (random intercept) and connects them with the associated error (CIs) (6,7).Those begging categories were then associated with the ID of each culled deer.

SARS-CoV-2 Surrogate Virus Neutralization Test
We performed SARS-CoV-2 surrogate virus neutralization tests on deer serum samples by using the Genscript cPass SARS-CoV-2 sVNT Kit (Genescript, https://www.genscript.com)according to the manufacturer's instructions.Fallow deer serum samples were thawed and heat inactivated at 56°C for 30 minutes in a water bath.Samples and controls were diluted 1:10 and mixed with horseradish peroxidase-labeled receptor-binding domain solution before incubating at 37°C for 30 minutes.Reaction mixtures were added to an ACE2-coated microtiter plate and incubated at 37°C for 15 minutes.The plate was washed and 3,3′,5,5′-tetramethylbenzidine substrate was added to each well, and the plate was incubated in the dark at 25°C for 15 minutes.
The reactions were then quenched with stop solution before immediately reading the samples on a Clariostar plate reader (BMG Labtech, https://www.bmglabtech.com).

Nucleic Acid Extraction and SARS-CoV-2 qRT-PCR
Twenty-five mg of each tissue sample was added to 1 mL of TRIzol with a single 5-mm stainless steel bead (QIAGEN, https://www.qiagen.com)and homogenized at maximum speed for 2 minutes by using a TissueLyser II (QIAGEN).Then, 0.1 mL of 1-bromo-3-chloropropane was added to each homogenized sample and shaken vigorously before incubating for 3 minutes.
The sample was spun in a precooled centrifuge (4°C, 12,000 × g for 15 minutes), after which 0.45 mL of the upper aqueous layer was transferred to a new tube and combined with 1 equivalent volume of 70% ethanol and mixed gently.The sample was added to RNeasy spin columns (QIAGEN) and the manufacturer's protocol was then followed.

Interface
Lungs from 2 seronegative fallow deer were removed immediately after culling.The tissues underwent gross and microscopic evaluation; no morphologic evidence of respiratory disease was observed in tissues used for ex vivo infections.One lung from each animal was clamped across the mainstem bronchus by using a hemostat and transported to the laboratory on infected with SARS-CoV-2 Italy-INMI-1 as a positive control and uninfected cell pellet as a negative control.The optimal antibody dilution was 1:1000 and incubation time was 30 minutes.Tissue sections were then washed and incubated with horse radish peroxidase from the EnVision Flex kit for 20 minutes.The chromogen 3,39-diaminobenzidine was used to visualize positive antibody staining (sections were incubated twice for 5 minutes each).Negative controls were run under identical conditions for each case by replacing the primary antibody with diluent.An isotype control (IHC universal negative control reagent; Enzo Life Sciences, https://www.enzo.com)was also included for each sample.Slides were counterstained with hematoxylin (Agilent Technologies Inc.) and rinsed in deionized water.Slides were dehydrated, permanently mounted, and then scanned and digitized by using the Aperio AT2 Digital Slide Scanner, and images were reviewed by using Aperio ImageScope 12.4 software (both Leica Biosystems).